ISOLATION OF WHITE SPOT SYNDROME VIRUS (WSSV) IN EGYPTIAN SHRIMP USING CONVENTIONAL PCR AND REAL TIME PCR (QPCR) TECHNIQUES

Yaser Hafez, Nader Moustafa, Asmaa Magouz, Naglaa Al-Maria

Abstract


Shrimp aquaculture industry threated by high mortality rates and severe economic losses as a result of white spot syndrome virus (WSSV) infection. Early-screening and diagnosis of WSSV are great strategies to decrease the economic losses of the disease on shrimp aquaculture. Therefore, this study was carried out to detect of white WSSV infected shrimp under using two molecular based methods, conventional PCR and qPCR. A total number of 90 samples of red (Aristeus antennatus) and gray (Penaeus latisclcatus) shrimp were collected from Kafr El-Sheikh and Alexandria governorates. External examination of shrimps collected from Kafr El-Sheikh Governorate revealed typical WSSV clinical signs (including loose and easily detached cuticle with appearance of small white spots (3 mm in diameter) and /or larger patches in the external surface of carapace and cephalothorax. The internal examination showed yellowish white, fragile and swollen hepatopancreas and swollen or shrunken lymphoid tissue. Red shrimp showed slightly obvious white spots without any internal lesions. PCR results confirmed the clinical investigation and postmortem (PM) examination and revealed presence of WSSV partial sequences with a size of 190 bp in shrimp samples from Kafr El-Sheikh Governorate.  In contrast, samples collected from Alexandria (Borg El-Arab) gave negative results. The result of qPCR confirmed that obtained by conventional PCR and showed that all positive results of WSSV by conventional PCR gave cycle threshold (Ct) values ranged from 34.81 to 40.06. Our results concluded that, WSSV Diseases of shrimp attack shrimp markets of Kafr El-Sheikh Governorate. The conventional PCR and qPCR based methods for isolation and identification of shrimp WSSV, provided accurate results.

Key words: WSSV; PCR; qPCR; shrimp


Full Text:

PDF

References


Shahidy MS, El-Gamal RM, Dessouki A. et al. Detection of white spot syndrome in cul-tured penaeid shrimp in Egypt: histopathologi-cal observation and polymerase chain reaction. MENA Science Journal 2015; 1(1): 5–14.

Tao M, Zhou H, Luo K, Lu J, Zhang Y, Wang F. Quantitative serum proteomics anal-yses reveal shrimp responses against WSSV infection. Dev Comp Immunol. 2019; 93:89–92.

Sharawy ZZ, Thiele R, Abbas EM, El-Magd MA, Hassaan MS, Peter C, Schmidt J, Saborowski R, Goda AMA-S, Slater MJ. Anti-oxidant response, body composition of whiteleg shrimp Litopenaeus vannamei co-cultured with Nile tilapia Oreochromis niloticus in recirculat-ing aquaculture. Aquaculture Environment In-teractions 2017; 9:257–68.

Pillai, N.G.K.; Katiha, P.K. Evolution of fisheries and aquaculture in India. Central Ma-rine Research Institute, Kochi, India 2004; p240.

FAO. Fishery and Aquaculture Statistics. FAO Year Book 2006. Food and Agriculture Organization of the United Nations, Rome 2008; p57.

Megahed ME, Kanrar S, Dhar AK . Com-plete Genome Sequence of White Spot Syn-drome Virus Isolated from Indian White Prawn (Fenneropenaeus indicus) in Egypt. Microbiol Resour Announc 2019; 3(1): 8–14.

Tsai Y, Lina Y, Choua P, et al. Detection of white spot syndrome virus by polymerase chain reaction performed under insulated iso-thermal conditions. Journal of Virological Methods 2012; 181(1):134–7.

Mayo, M. A. A summary of taxonomic changes recently approved by ICTV. Archives of Virology2002; 147, 1655–63.

Van Hulten M, Witteveldt J, Peters S. et al. The white spot syndrome virus DNA genomic sequence. Virology 2001; 286: 7–22.

Yang F, He J, Lin X, et al. Complete ge-nome sequence of the shrimp white spot bacilli-form virus. Journal of Virology 2001; 75: 11811–20.

Vaseeharan B, Jayakumar R, and Rama-samy, A. PPCR-based detection of white spot syndrome virus in cultured and captured crusta-ceans in India. Lett. Appl. Microbiol 2003; 37: 443–7.

Jian X, Lu L, Chen Y, et al. Comparison of a novel in situ polymerase chain reaction (ISPCR) method to other methods for white spot syndrome virus (WSSV) detection in Pe-naeus vannamei. Dis. Aquat. Organ 2005; 67: 171–6.

Durand S, and Lightner D. Quantitative real-time PCR for the measurement of white spot syndrome virus in shrimp. J. Fish Dis.2002; 25: 381–9.

Nunan, L, and Lightner D. Optimized PCR assay for detection of white spot syn-drome virus (WSSV). J. Virol. Methods 2011; 171: 318–21.

Chou P, Lin Y, Teng P, et al. Real-time target-specific detection of loop-mediated iso-thermal amplification for white spot syndrome virus using fluorescence energy transfer-based probes. J. Virol. Methods 2011; 173: 67–74.

Kono T, Savan R, Sakai M, Itami T. De-tection of white spot syndrome virus in shrimp by loop-mediated isothermal amplification. J. Virol. Methods 2004; 115: 59–65.

Pradeep B, Karunasagar I and Karunasa-gar I. Fitness and virulence of different strains of white spot syndrome virus. Journal of Fish Diseases 2009; 32: 801–5.

Flegel TW. Detection of major penaeid shrimp viruses in Asia, a historical perspective with emphasis on Thailand. Aquaculture 2006; 258: 1–33.

Eissa I, Badran A, Diab A, et al. Diagno-sis of white spot syndrome virus (WSSV) among shrimp for the first time in Egypt. SCVMJ, VIX 2009; (1).

Orabi A, Hussein A, Saleh A A, El-Magd M A, Munir M. Evolutionary insights into the fusion protein of Newcastle disease virus iso-lated from vaccinated chickens in 2016 in Egypt, Archives of Virology 2017; 162(10): 3069–79.

Lightner DV. A handbook of pathology and diagnostic procedures for diseases of pe-naeid shrimp. Baton Rouge, Louisiana, USA: World Aquaculture Society1996.

Salama A, Diab A, Abd El-Samie, A, and Abdel-Wahab A. Isolation and Identification of White Spot Syndrome Virus. Zag. Vet. J. 2008; (ISSN. 1110-1458) Vol.36, No. 4 pp.105–13.

Wang Y, Lee K, Najiah M. et al. A new bacterial white spot syndrome (BWSS): in cul-tured tiger shrimp Penaeus monodon and its comparison with white spot syndrome (WSS): caused by virus. Dis. Aquat. Org.2002; 41 (1): 9–18.

Lotz J, and Soto M A. Model of white spot syndrome virus (WSSV) epidemics in Li-topenaeus vannamei. Diseases of Aquatic Or-ganisms 2002; 50: 199–209.

Wu W, Wang L and Zhang X. Identifica-tion of white spot syndrome virus (WSSV) en-velope proteins involved in shrimp infection. Virology 2005; 332: 578–83.

Chen L, Leu J, Huang C. et al. Identifica-tion of a nucleocapsid protein (VP35) gene of shrimp white spot syndrome virus and charac-terization of the motif important for targeting VP35 to the nuclei of transfected insect cells. Virology 2002; 293: 44–53.

Wang Q, White B, Redman R and Light-ner D. Per os challenge of Litopenaeus van-namei postlarvae and Farfantepenaeus duorarum juveniles with six geographic isolates of white spot syndrome virus. Aquaculture 1999; 170: 179–194.

Kasornchandra J, Boonyaratpalin S, and Itami T. Detection of white spot syndrome in cultured penaeid shrimp in Asia: microscopic observation and polymerase chain reaction. Aquaculture 1998; 164: 243–51.

Peng S, Lo C, Ho C et al. Detection of white spot baculovirus (WSBV) in giant fresh water prawn; Macrobrachium rosenbergii using polymerase chain reaction. Aquaculture 1998; 164: 253–62.

Karunasagar I, Otta S and Karunasagar I. Histopathological and bacteriological study of white spot syndrome of Penaeus monodon along the west coast of India. Aquaculture 1997; 153: 9–13.

Chang P, Lo C, Wang Y, and Kou G. Identification of white spot syndrome virus associated baculovirus (WSBV) target organs in the shrimp Penaeus monodon by in situ hybrid-ization. Diseases of Aquatic Organisms 1996; 27: 131–9.

Lo C, Leu J, Ho C, et al. Detection of baculovirus associated with white spot syn-drome (WSBV) in penaeid shrimps using poly-merase chain reaction. Dis. Aquat. Org.1996; 25: 133–41.

Wang Y, Lo C, Chang P and Kou G. Ex-perimental infection of white spot baculovirus in some cultured and wild decapods in Taiwan. Aquaculture 1998; 164: 221–31.

Tang KFJ. Lightner, D.V. Quantification of white spot syndrome virus DNA through a compe-

titive polymerase chain reaction. Aquaculture 2000; 189: 11–21.

Kathy Tang-Nelson and Donald V. Lightner. Development of real-time PCR assays for detection of white spot syndrome virus, yellow head virus, Taura syndrome virus, and infectious hypodermal and hematopoietic ne-crosis virus in penaeid shrimp 2001, NA06FD0448.




DOI: http://dx.doi.org/10.26873/SVR-783-2019

Refbacks

  • There are currently no refbacks.


SLOVENIAN VETERINARY RESEARCH, Veterinary Faculty
Gerbičeva 60, SI-1000 Ljubljana, Slovenia, T: +386 (0)1 47 79 100, F: +386 (0)1 28 32 243, E: slovetres@vf.uni-lj.si
Published by computing.si